Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Characterisation of peptide interactions that regulate protein kinase Cε activation (#283)

Dorothy C. Wai 1 , Indu R. Chandrashekaran 1 , Carsten Schmitz-Peiffer 2 , Raymond S. Norton 1
  1. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia
  2. Diabetes and Metabolism Division, Garvan Institute of Medical Research, University of New South Wales, Sydney, NSW, Australia

Protein kinase C (PKC) isoforms are involved in diverse signalling pathways, and aberrant activity of PKCs has been implicated in pathological states, including cancer, autoimmunity, ischaemia and diabetes [1]. In contrast with the highly-conserved ATP- and substrate-binding sites in the C-terminal kinase domain, the N-terminal C2 domain in some PKC isoforms interacts with distinct regulatory proteins, thus providing a potential means for isoform-specific targeting by small molecules.

PKCε is associated with insulin resistance in type II diabetes [2]. Upon activation of PKCε, the PKCε C2 domain is bound by the scaffolding protein Receptor for Activated C-Kinase 2 (RACK2), leading to translocation of PKCε to the cell membrane and inhibition of insulin receptor kinase activity. A putative RACK2-binding site in PKCε C2, designated εV1-2, has been identified as an antagonist of PKCε translocation [3]. It has also been observed that a site within PKCε C2, pseudoεRACK (ψεRACK), bears some similarity to a sequence within RACK2. A ψεRACK peptide has been shown to enhance PKCε translocation in vivo, leading to the proposition that an intramolecular interaction between the ψεRACK and εV1-2 sites stabilises the inactive form of PKCε [4]. Characterisation of these regulatory interactions would facilitate development of PKCε-selective inhibitors.

In the present work, direct interactions between PKCε, RACK2 and εV1-2/ψεRACK peptides are tested by SPR and NMR. The binding of full-length RACK2 to the εV1-2 peptide and inhibition of the PKCε-RACK2 interaction by εV1-2 are confirmed. We further demonstrate that the PKCε C2 domain participates in an intramolecular interaction with full-length PKCε, but none of the εV1-2, ψεRACK or RACK2-derived peptides are able to bind PKCε. These results support the involvement of the εV1-2 site in the PKCε-RACK2 interaction, and indicate that further work is required to determine the PKCε-binding site of RACK2 and the site(s) involved in PKCε intramolecular interactions.

  1. D. Mochly-Rosen, K. Das, and K.V. Grimes, Nat. Rev. Drug Discov. 11, 12 (2012).
  2. C. Schmitz-Peiffer and T.J. Biden, Diabetes. 57, 7 (2008).
  3. J.A. Johnson, et al., J. Biol. Chem. 271, 40 (1996).
  4. G.W. Dorn, et al., P. Natl Acad. Sci. 96, 22 (1999).